WO2016072448A1 - Endoxylanase mutante, composition enzymatique utilisable en vue de la décomposition d'une biomasse et procédé de production d'une solution de sucre - Google Patents

Endoxylanase mutante, composition enzymatique utilisable en vue de la décomposition d'une biomasse et procédé de production d'une solution de sucre Download PDF

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WO2016072448A1
WO2016072448A1 PCT/JP2015/081151 JP2015081151W WO2016072448A1 WO 2016072448 A1 WO2016072448 A1 WO 2016072448A1 JP 2015081151 W JP2015081151 W JP 2015081151W WO 2016072448 A1 WO2016072448 A1 WO 2016072448A1
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endoxylanase
amino acid
seq
acid sequence
mutant
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PCT/JP2015/081151
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English (en)
Japanese (ja)
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栗原　宏征
拓也 笠原
千晶 山田
奈津子 村上
山田　勝成
石川　一彦
真宏 渡邊
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東レ株式会社
国立研究開発法人産業技術総合研究所
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Priority to CA2966330A priority Critical patent/CA2966330A1/fr
Priority to AU2015344324A priority patent/AU2015344324B2/en
Priority to JP2016557794A priority patent/JP6689487B2/ja
Priority to BR112017008702-2A priority patent/BR112017008702A2/pt
Priority to EP15856688.5A priority patent/EP3216864A4/fr
Priority to US15/521,654 priority patent/US10435728B2/en
Publication of WO2016072448A1 publication Critical patent/WO2016072448A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a novel endoxylanase mutant, an enzyme composition for decomposing biomass containing the same, and a method for producing a sugar solution.
  • cellulose-containing biomass contains, in addition to cellulose, hemicelluloses such as xylan and arabinan, and lignin.
  • Xylan has ⁇ -1,4-linked D-xylose as the main chain, and O-acetyl, ⁇ -arabinofuranosyl, glucuronic acid, and phenolic acid are partially modified to this main chain. (Non-patent Document 1).
  • Xylanase is an important enzyme that degrades cellulose-containing biomass by acting on a ⁇ -1,4-linked xylose backbone.
  • Xylanases are classified into Family 10 (GH10) and Family 11 (GH11) based on amino acid sequence homology (Non-patent Document 2).
  • GH10 xylanase generally has a molecular weight of 30 kDa or more, whereas GH11 xylanase is generally said to have a molecular weight of about 20 kDa (Non-patent Document 3).
  • Filamentous fungi are known as microorganisms that degrade a wide variety of cellulosic biomass.
  • Cellulase produced by Acremonium cellulolyticus in the culture solution is known to obtain a higher glucose yield than the cellulase produced by Trichoderma reesei in the degradation of cellulose-containing biomass (Non-Patent Document) 4).
  • Non-Patent Document 5 seven xylanases have been cloned from Acremonium cellulolyticus, and functional analysis of the wild-type enzyme has been carried out.
  • XylC is reported to possess the highest xylan-degrading activity although it is the least expressed in Acremonium cellulolyticus.
  • An object of the present invention is to provide a novel endoxylanase mutant with improved thermostability. Moreover, it is providing the enzyme agent containing this and the manufacturing method of an efficient sugar liquid.
  • the present inventors have found that the amino acid sequence of filamentous fungal endoxylanase has positions 35, 44, 62, 63, and 101 of the amino acid sequence of SEQ ID NO: 1.
  • the present inventors have found that the thermal stability of the endoxylanase can be improved by substituting an amino acid residue at a position corresponding to one or more positions selected from position 102 with another amino acid residue, and completed the present invention I came to let you. That is, the present invention has the following configuration.
  • An endoxylanase variant comprising an amino acid sequence in which the amino acid residues are substituted and having endoxylanase activity.
  • the amino acid residue at a position corresponding to position 44 and / or position 63 of the amino acid sequence of SEQ ID NO: 1 is from histidine, glycine, tryptophan, methionine, proline, alanine, phenylalanine, valine, leucine, and isoleucine,
  • the amino acid residue at a position corresponding to position 101 and / or position 102 of the amino acid sequence of SEQ ID NO: 1 is substituted with any amino acid independently selected from proline and asparagine The endoxylanase mutant of any one of [1] to [3].
  • any one of [1] to [4], wherein one or more amino acid residues selected from positions corresponding to position 61, position 65, and position 66 of the amino acid sequence of SEQ ID NO: 1 are substituted An endoxylanase variant.
  • the amino acid residues at positions corresponding to position 35, position 44, position 61, position 62, position 63, position 65, position 66, position 101, and position 102 of the amino acid sequence of SEQ ID NO: 1 are all substituted.
  • [7] The endoxylanase mutant of any one of [1] to [6], wherein the filamentous fungus-derived endoxylanase is derived from Acremonium cellulolyticus.
  • An expression vector comprising the DNA of [9].
  • a transformed cell produced by transformation using the expression vector of [10].
  • a method for producing an endoxylanase mutant comprising culturing the transformed cell of [11] and obtaining an endoxylanase mutant produced by the transformed cell.
  • An biomass decomposing enzyme composition comprising the endoxylanase mutant of any one of [1] to [8].
  • the enzyme composition is one selected from the group consisting of cellobiohydrolase, endoglucanase, ⁇ -glucosidase, ⁇ -xylosidase, mannanase, mannosidase, glucoamylase, ⁇ -amylase, esterase, and lipase.
  • the enzyme composition for decomposing biomass according to [13] further comprising two or more enzymes.
  • a method for producing a sugar solution from the biomass comprising adding the enzyme composition for decomposing biomass according to [13] or [14] to the biomass.
  • an endoxylanase mutant with improved thermostability can be provided.
  • the endoxylanase mutant of the present invention has the effect that the thermal stability at high temperature conditions, specifically 65 ° C. or higher, is improved, and the xylan decomposition activity is also improved. Therefore, the endoxylanase mutant of the present invention and the enzyme composition containing the endoxylanase mutant of the present invention can be suitably used for producing a sugar solution from cellulose-containing biomass.
  • FIG. 1 is a photograph showing the results of SDS-PAGE of the culture supernatant of yeast Pichia pastoris expressing wild-type endoxylanase or any of the endoxylanase mutants.
  • M is a marker
  • 1 is a wild-type endoxylanase
  • 2 is an endoxylanase variant containing substitutions at positions 35 and 62
  • 3 is position 35, position 44, position 62, position 61, position 65, position 63.
  • Endoxylanase mutant is an enzyme having an activity of hydrolyzing hemicellulose (endoxylanase activity) by acting on a ⁇ -1,4-linked xylose main chain. It is an enzyme classified in the number 3.3.1.8. Endoxylanases are classified into two types, family 10 (GH10) and family 11 (GH11). The endoxylanase of the present invention is an enzyme classified into family 11 (GH11), and has a ⁇ -jelly roll structure. Possess.
  • the “endoxylanase activity” can be measured using ⁇ -1,4-linked D-xylose as a substrate, preferably Birchwood xylan sold as a reagent as a substrate. Whether or not xylan as a substrate has been decomposed can be confirmed by measuring the amount of reducing sugar contained in the reaction solution after the reaction.
  • the amount of reducing sugar can be measured by using the dinitrosalicylic acid method (DNS method), and Bailey et al. “Interlaboratory testing of methods for xylanase activity” J. Am. Biotechnol. 23, 257-270 can be preferably used.
  • the conditions for measuring the activity are not particularly limited as long as the activity of endoxylanase can be measured by the method described above.
  • a range of 20 ° C. to 90 ° C. preferably a range of 40 ° C. to 75 ° C.
  • a pH of 4 to 9 is preferable
  • a pH of 5 to 7 is more preferable
  • a reaction time is It is preferably 1 second to 600 minutes, and most preferably 1 minute to 60 minutes.
  • the xylan used as a substrate at the time of activity measurement is preferably in the range of 0.1% by weight to 10% by weight, and most preferably in the range of 0.5% by weight to 2% by weight.
  • an endoxylanase can be used that is derived from a filamentous fungus, such as Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces. ), Humicola, Acremonium, Irpex, Mucor, Talaromyces, and the like, preferably those derived from the genus Acremonium.
  • the endoxylanase isolated from Acremonium cellulolyticus can be particularly preferably used as the endoxylanase of the present invention.
  • the endoxylanase of Acremonium cerulolyticus is known. For example, it is registered in GenBank etc. as AB874990, AB8749991, AB8749992, AB874993, AB874994, AB874949, AB874949, and these gene information is used in the present invention. be able to.
  • the endoxylanase preferably includes the amino acid sequence represented by SEQ ID NO: 1 or consists of the amino acid sequence.
  • the endoxylanase includes a part of the endoxylanase as long as the endoxylanase activity is maintained.
  • “part of endoxylanase” means at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the original endoxylanase activity.
  • it consists of a fragment of endoxylanase from which any partial region has been removed. Examples of such a fragment include those obtained by removing the signal peptide region from endoxylanase.
  • the signal peptide include a region represented by the amino acid sequence from position 1 to position 34 in the amino acid sequence represented by SEQ ID NO: 1.
  • a part of the endoxylanase includes the amino acid sequence represented by SEQ ID NO: 2 or consists of the amino acid sequence.
  • the “endoxylanase variant” of the present invention corresponds to position 35, position 44, position 62, position 63, position 101, and position 102 of the amino acid sequence of SEQ ID NO: 1 in the amino acid sequence of “endoxylanase” described above. It means a protein having an endoxylanase activity in which amino acid residues at one or more positions selected from positions are substituted with another amino acid. More preferably, the endoxylanase variant of the present invention has two, three, or three selected from positions corresponding to position 35, position 44, position 62, position 63, position 101, and position 102 of the amino acid sequence of SEQ ID NO: 1. Contains 4, 4, 5 or 6 amino acid substitutions. Particularly preferably, the endoxylanase variant of the invention comprises amino acid substitutions at all six positions corresponding to position 35, position 44, position 62, position 63, position 101 and position 102 of the amino acid sequence of SEQ ID NO: 1. .
  • the “endoxylanase mutant” of the present invention has higher heat resistance than the endoxylanase not containing the amino acid substitution by including the amino acid substitution at the above position.
  • amino acid positions specified by “positions corresponding to position 35, position 44, position 62, position 63, position 101, and position 102 of the amino acid sequence of SEQ ID NO: 1” are as follows: It can be determined by a method including procedures 1) to 3).
  • Procedure 2 Next, the amino acid positions corresponding to position 35, position 44, position 62, position 63, position 101, and position 102 of the amino acid sequence represented by SEQ ID NO: 1 in the amino acid sequence of the above endoxylanase are determined. .
  • the corresponding amino acid position can be determined by aligning the amino acid sequence of the endoxylanase with the amino acid sequence of SEQ ID NO: 1. Such an operation is called amino acid sequence alignment.
  • the alignment tool many well-known software such as ClustalW is used, and default parameters are used.
  • amino acid position corresponding to position 102 can be determined.
  • the amino acid substitution at each position is not particularly limited as long as it is a substitution with another amino acid, and preferably includes substitution with the following amino acids: Position 35: cysteine; Position 44: histidine, glycine, tryptophan, methionine, proline, alanine, phenylalanine, valine, leucine or isoleucine, preferably histidine; Position 62: cysteine; Position 63: histidine, glycine, tryptophan, methionine, proline, alanine, phenylalanine, valine, leucine or isoleucine, preferably leucine; Position 101: proline, or asparagine, preferably proline; Position 102: Proline or asparagine, preferably asparagine.
  • amino acids at positions corresponding to positions 35 and 62 are both substituted with cysteine, whereby a disulfide bond can be formed on the cysteine side chain at the position.
  • the amino acids at the positions corresponding to position 101 and position 102 may remain as wild-type amino acids without being substituted.
  • the positions corresponding to positions 101 and 102 may be included in the amino acid sequence that undergoes sugar chain modification in eukaryotes.
  • the “endoxylanase variant” of the present invention includes amino acid substitution at a position selected from positions corresponding to position 35, position 44, position 62, position 63, position 101, and position 102 of the amino acid sequence of SEQ ID NO: 1.
  • amino acid substitution at one or more positions selected from position 61, position 65, and position corresponding to position 66 of the amino acid sequence of SEQ ID NO: 1 may be included.
  • the heat resistance can be further increased.
  • the endoxylanase variant of the present invention further comprises two or three amino acid substitutions selected from position 61, position 65 and the position corresponding to position 66 of the amino acid sequence of SEQ ID NO: 1. More preferably, the endoxylanase variant of the present invention further comprises amino acid substitutions at three positions corresponding to position 61, position 65, and position 66 of the amino acid sequence of SEQ ID NO: 1.
  • the amino acid substitution at each position may be any substitution with another amino acid, and is not particularly limited, but includes substitution with the following amino acids: Position 61: glycine, tryptophan, methionine, proline, alanine, phenylalanine, valine, leucine or isoleucine, preferably methionine; Position 65: proline; Position 66: Glycine, tryptophan, methionine, proline, alanine, phenylalanine, valine, leucine or isoleucine, preferably glycine.
  • the endoxylanase variant of the present invention has one or more positions selected from position 35, position 44, position 62, position 63, position 101, and position 102 in the amino acid sequence of SEQ ID NO: 1.
  • a polypeptide having or having an endoxylanase activity having an amino acid sequence in which the amino acid is substituted or a part thereof. Examples of the “part thereof” include polypeptides from which the signal peptide region has been removed.
  • endoxylanase variants include the following: An endoxylanase variant containing a substitution for cysteine at position 35, comprising or consisting of the amino acid sequence of SEQ ID NO: 3 (the amino acid sequence of SEQ ID NO: 3 contains the region of the signal peptide) Absent); An endoxylanase variant containing a substitution to cysteine at position 62, comprising or consisting of the amino acid sequence of SEQ ID NO: 5 (the amino acid sequence of SEQ ID NO: 5 contains the region of the signal peptide) Absent); An endoxylanase variant containing a substitution to cysteine at both positions 35 and 62, and comprising or consisting of the amino acid sequence of SEQ ID NO: 9 (the amino acid sequence of SEQ ID NO: 9 contains the above signal Does not include the peptide region); An endoxylanase variant comprising a substitution for histidine at position 44, comprising or consisting of the amino acid sequence of SEQ ID NO: 4 (the amino acid sequence of SEQ ID
  • the endoxylanase variant of the invention has a substitution in the amino acid sequence of SEQ ID NO: 1 in addition to a substitution of a position selected from position 35, position 44, position 62, position 63, position 101, and position 102.
  • the endoxylanase variant of the present invention has the amino acid sequence of SEQ ID NO: 1 at seven positions of position 35, position 44, position 62, position 63, position 61, position 65, and position 66.
  • the polypeptide includes or consists of a polypeptide having an amino acid sequence substituted with an amino acid or a part thereof and having endoxylanase activity. More specifically, such an endoxylanase variant includes or consists of the amino acid sequence of SEQ ID NO: 45 (the amino acid sequence of SEQ ID NO: 45 does not include the signal peptide region).
  • the endoxylanase variant of the present invention has a position 35, position 44, position 62, position 63, position 101, position 102, position 61, position 65, and position in the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide comprises or consists of a polypeptide having an amino acid sequence in which amino acids at 9 positions of 66 are substituted or a part thereof and having endoxylanase activity. More specifically, such an endoxylanase mutant includes or consists of the amino acid sequence of SEQ ID NO: 10 (the amino acid sequence of SEQ ID NO: 10 does not include the signal peptide region).
  • the endoxylanase mutant of the present invention includes the amino acid position 35, position 44, position 61, position 62, position 63, position 65, position 66, position 101 in the amino acid sequence of the endoxylanase mutant or a part thereof. Also included are proteins in which the substituted amino acid at position 102 is not mutated, has a deletion, substitution, addition or insertion of one or several amino acids and has endoxylanase activity.
  • the range of “1 or several” is not particularly limited, but is, for example, within 10 pieces, more preferably within 5 pieces, particularly preferably within 4 pieces, or 1 piece or 2 pieces.
  • the endoxylanase mutant of the present invention also has the amino acid position 35, position 44, position 61, position 62, position 63, position 65, position 66, position in the amino acid sequence of the endoxylanase mutant or a part thereof.
  • 101 and the amino acid substituted at position 102 is not mutated, and except for the amino acid, the above endoxylanase mutant or a part of the amino acid sequence thereof and BLAST ((Basic Local Alignment Tool National Center for Biological) Information (basic local alignment search tool of the National Center for Biological Information)) etc.
  • identity refers to identical amino acids and similarities to all overlapping amino acid residues in an optimal alignment when two amino acid sequences are aligned with or without introducing a gap. It means the percentage of amino acid residues.
  • sequence analysis software for example, known algorithms such as BLAST and FASTA.
  • the endoxylanase mutant of the present invention may have an additional peptide or protein added to the N-terminus and / or C-terminus.
  • additional peptide or protein examples include those containing methionine as a translation initiation point, secretory signal sequence, transport protein, binding protein, tag peptide for purification, heterologous hydrolase, fluorescent protein and the like.
  • those skilled in the art can select a peptide or protein having a function to be added according to the purpose and add it to the endoxylanase mutant of the present invention.
  • the endoxylanase mutant of the present invention is prepared, for example, by preparing DNA encoding the amino acid sequence of the endoxylanase mutant described in (1) above, and ligating it to an expression vector. It can be produced by introducing an expression vector into a host, producing it as a heterologous or homologous protein, and isolating and purifying it.
  • the codon usage frequency encoding the amino acid sequence may be the same as the filamentous fungus derived from endoxylanase, for example, Acremonium cellulolyticus (Acremonium cellulolyticus), or may be changed according to the codon usage frequency of the host. Good.
  • a conventionally known method can be used as a method for preparing the DNA encoding the above-mentioned endoxylanase mutant. For example, a method of totally synthesizing a DNA encoding a target amino acid sequence by gene synthesis, or a filamentous form A DNA encoding an endoxylanase isolated from a bacterium or a part thereof is mutated so that the DNA encoding the amino acid at the predetermined position encodes another predetermined amino acid by site-directed mutagenesis. The method etc. which introduce
  • transduce are mentioned.
  • a site-specific mutagenesis method for causing mutation at a target site of DNA it can be carried out by a conventional and commonly used PCR method.
  • DNA encoding the endoxylanase of the present invention it is particularly preferable to use a DNA encoding endoxylanase isolated from Acremonium cellulolyticus.
  • DNA encoding endoxylanase encodes a signal peptide from DNA comprising the base sequence represented by SEQ ID NO: 32, DNA comprising the base sequence, or the base sequence represented by SEQ ID NO: 32 DNA containing the base sequence represented by SEQ ID NO: 33 from which the region to be removed has been removed or DNA comprising the base sequence can be used.
  • These DNAs can be obtained by isolating DNA from Acremonium cellulolyticus according to a known method and amplifying the DNA by a technique such as PCR.
  • the DNA encoding the endoxylanase variant of the present invention is 1 or 2 selected from amino acid position 35, position 44, position 62, position 63, position 101, and position 102 in the amino acid sequence of SEQ ID NO: 1. It contains or consists of a DNA encoding a polypeptide having an amino acid sequence in which amino acids in the above positions are substituted or a part thereof and having endoxylanase activity.
  • the DNA encoding such an endoxylanase variant includes the following: DNA encoding an endoxylanase variant containing a substitution for cysteine at position 35, comprising or consisting of the base sequence of SEQ ID NO: 34 (the DNA does not encode the signal peptide region) ); DNA encoding an endoxylanase variant containing a cysteine substitution at position 62 and comprising or consisting of the base sequence of SEQ ID NO: 36 (the DNA does not encode the signal peptide region) ); DNA encoding an endoxylanase mutant containing a substitution to cysteine at both positions 35 and 62, and comprising the base sequence of SEQ ID NO: 40 or consisting of the base sequence (the DNA comprises the signal peptide Is not coded)); DNA encoding an endoxylanase variant containing a substitution for histidine at position 44 and comprising or consisting of the base sequence of SEQ ID NO: 35 (the DNA does not encode the region of the signal peptide)
  • the DNA encoding the endoxylanase variant of the present invention has a substitution at position 61 in addition to the four substitutions at positions 35, 44, 62 and 63 described above in the amino acid sequence of SEQ ID NO: 1.
  • the DNA encoding such an endoxylanase mutant includes the base sequence of SEQ ID NO: 49 or consists of the base sequence (the DNA does not encode the signal peptide region).
  • the DNA encoding the endoxylanase variant of the present invention has six substitutions at position 35, position 44, position 62, position 63, position 101 and position 102 described above in the amino acid sequence of SEQ ID NO: 1.
  • it has an amino acid sequence including a substitution at position 61 with methionine, a substitution at position 65 with aspartic acid, and a substitution at position 66 with asparagine, or a part thereof, and has endoxylanase activity
  • the DNA encoding such an endoxylanase mutant includes the base sequence of SEQ ID NO: 41 or consists of the base sequence (the DNA does not encode the signal peptide region).
  • the DNA encoding the endoxylanase mutant of the present invention includes amino acids substituted at amino acid position 35, position 44, position 61, position 62, position 63, position 65, position 66, position 101, and position 102.
  • DNA containing the following base sequence or DNA consisting of the base sequence is also included: A base sequence in which one or more bases are deleted, substituted, added or inserted in the above base sequence.
  • SEQ ID NO: 1 1 to 100 bases, preferably 1 to 50 bases, more preferably 1 to 10 bases are deleted, substituted, added or inserted.
  • Sequence A nucleotide sequence having 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 99% or more of the above nucleotide sequence. Comparison of base sequences can be performed by a known method, for example, BLAST or the like can be performed using, for example, default settings; A base sequence that hybridizes under stringent conditions with DNA comprising a sequence complementary to the above base sequence. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, 2 to 6 ⁇ SSC (composition of 1 ⁇ SSC: 0.15M NaCl, 0 Hybridization is performed at 42 to 55 ° C.
  • a DNA encoding the endoxylanase mutant prepared as described above is ligated downstream of a promoter in an appropriate expression vector using a restriction enzyme and DNA ligase to produce an expression vector containing the DNA. Can do.
  • expression vectors include bacterial plasmids, yeast plasmids, phage DNA (such as lambda phage), retrovirus, baculovirus, vaccinia virus, adenovirus and other viral DNA, SV40 derivatives, and other Agrobacterium as a vector for plant cells. Any other vector can be used as long as it can replicate and survive in the host cell. For example, when the host is E. coli, pUC, pET, pBAD and the like can be exemplified.
  • pPink-HC When the host is yeast, pPink-HC, pPink-LC, pPink ⁇ -HC, pPCIZ, pPCIZ ⁇ , pPCI6, pPCI6 ⁇ , pFLD1, pFLD1 ⁇ , pGAPZ, pGAPZ ⁇ , pPIC9K, pPIC9, pD912, pD915, etc.
  • the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
  • the host is Escherichia coli
  • lac promoter, Trp promoter, PL promoter, PR promoter and the like are used.
  • AOX1 promoter, TEF1 promoter, ADE2 promoter, CYC1 promoter, GAL-L1 promoter, GAP promoter and the like can be mentioned. It is done.
  • the host cells used in the present invention are preferably Escherichia coli, bacterial cells, yeast cells, fungal cells, insect cells, plant cells, animal cells and the like.
  • yeast cells include the genus Pichia, the genus Saccharomyces, and the genus Schizosaccharomyces.
  • fungal cells include Aspergillus and Trichoderma. Insect cells include Sf9, plant cells include dicotyledonous plants, and animal cells include CHO, HeLa, HEK293, and the like.
  • the host used in the present invention is preferably a eukaryotic microorganism, more preferably a yeast cell or a fungal cell. When yeast cells or fungal cells are used as a host, there may be advantages such that the enzyme production is large, the enzyme can be secreted and produced outside the cell, and / or the heat resistance of the enzyme can be increased.
  • Transformation or transfection can be performed by a known method such as a calcium phosphate method or an electroporation method.
  • the endoxylanase mutant of the present invention can be obtained by expressing the product in the host cell transformed or transfected as described above under the control of a promoter and recovering the product.
  • transformed or transfected host cells are propagated or grown to an appropriate cell density and then chemically induced means such as temperature shift or addition of isopropyl-1-thio- ⁇ -D-galactoside (IPTG)
  • IPTG isopropyl-1-thio- ⁇ -D-galactoside
  • the promoter is induced by and the cells are further cultured for a period of time.
  • the promoter can be induced by the sugar contained in the medium, and the cells can be cultured and expressed simultaneously.
  • endoxylanase mutant When the desired endoxylanase mutant is excreted outside the cell, it is directly from the medium, and when it is outside the cell, physical means such as ultrasonic disruption or mechanical disruption, or a cell lysing agent, etc.
  • the endoxylanase mutant is purified after cell destruction by chemical means.
  • endoxylanase variants can be obtained from recombinant cell culture using ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography, gel filtration chromatography. It can be partially or completely purified by combining techniques such as electrophoresis.
  • the biomass enzyme composition of the present invention contains at least the endoxylanase mutant of the present invention as an active ingredient for hydrolyzing biomass, and decomposes biomass. It is an enzyme composition used for a use.
  • the biomass refers to a biological plant, and examples thereof include herbaceous plants, woody plants, algae, seaweeds, sugar-producing crops, resource crops, and grains. These biomasses all contain disaccharides or more, and can be hydrolyzed by the enzyme composition for biomass decomposition of the present invention.
  • Cellulose-containing biomass can be particularly preferably used.
  • Cellulose-containing biomass is a biological resource containing a cellulose component.
  • herbaceous biomass such as bagasse, switchgrass, napiergrass, Eliansus, corn stover, rice straw and wheat straw, or woody biomass such as trees and waste building materials, algae, seaweed, etc. It refers to biomass.
  • cellulosic biomass contains lignin, which is an aromatic polymer, in addition to cellulose and hemicellulose (hereinafter referred to as “cellulose” as a generic term for cellulose and hemicellulose).
  • the endoxylanase mutant contained in the biomass decomposing enzyme composition of the present invention can be used either purified or roughly purified.
  • the endoxylanase mutant contained in the enzyme composition for biomass degradation of the present invention may be immobilized on a solid phase.
  • the solid phase include, but are not limited to, polyacrylamide gel, polystyrene resin, porous glass, and metal oxide.
  • the endoxylanase mutant of the present invention is advantageous in that it can be used continuously and repeatedly by being immobilized on a solid phase.
  • a processed product of cells transformed with DNA encoding the endoxylanase mutant can also be used as a crudely purified endoxylanase mutant.
  • the “processed product of transformed cells” includes transformed cells immobilized on a solid phase, killed and disrupted transformed cells, and those obtained by immobilizing them on a solid phase.
  • the enzyme composition for degrading biomass of the present invention may contain other enzymes in addition to the endoxylanase mutant of the present invention.
  • a hydrolase related to biomass degradation is preferably included.
  • examples of such other enzymes include cellobiohydrolase, endoglucanase, ⁇ -glucosidase, ⁇ -xylosidase, mannanase, mannosidase, glucoamylase, ⁇ -amylase, esterase, lipase, and the like.
  • These other enzymes are preferably enzymes produced by microorganisms such as filamentous fungi.
  • filamentous fungi include Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicora, and Humicola.
  • microorganisms such as the genus Irpex (Irpex), the genus Mucor (Mucor), and the genus Talaromyces (Talaromyces). Since these microorganisms produce an enzyme in the culture solution, the culture solution may be used as an unpurified enzyme as it is, together with the endoxylanase mutant of the present invention, to form the enzyme composition of the present invention.
  • the purified and formulated product may be combined with the endoxylanase mutant of the present invention to form the enzyme composition of the present invention.
  • the filamentous fungus for producing the other enzymes is preferably a filamentous fungus derived from the genus Trichoderma.
  • a cellulase mixture derived from Trichoderma reesei can be more preferably used.
  • Trichoderma reesei QM9414 Trichoderma reesei QM9414
  • Trichoderma reesei QM9123 Trichoderma reesei QM9123
  • Trichoderma reeseiRutC-30er Tricoderma reesei RutC-30er
  • Trichoderma reesei PC3-7 Trichoderma reesei CL-847 (Trichoderma reesei CL-847), Trichoderma reesei MCG77 (Trichoderma reesei MCG77), Trichoderma reesei MCGer 80
  • Cellulase mixtures thereof derived from dermatan viride QM9123 Trichoderma viride QM9123
  • it may be a mutant strain derived from the genus Trichoderma and subjected to a mutation treatment with a mutation agent or ultraviolet
  • the enzyme composition for decomposing biomass of the present invention may be added with substances other than enzymes, such as protease inhibitors, dispersants, dissolution promoters, stabilizers, buffers, preservatives, and the like.
  • the enzyme composition for decomposing biomass of the present invention can be used in a method for producing a sugar solution by adding it to biomass.
  • the sugar solution as used herein refers to a solution containing a saccharide obtained by hydrolyzing at least a polysaccharide derived from biomass into a saccharide having a lower molecular weight.
  • the sugar component in the sugar solution include xylose, glucose, cellobiose, xylobiose, xylotriose, xylotetraose, xylopentaose, mannose, arabinose, sucrose, fructose and the like.
  • the sugar solution obtained using the enzyme composition contains xylose, xylobiose, xylotriose, xylotetraose, xylopenta. Often contains aus.
  • the biomass used in the production of the sugar solution may be any biomass as long as it is described above.
  • a biomass pretreated for the purpose of increasing the sugar yield from the biomass can be used. Pretreatment refers to partially decomposing lignin and hemicellulose using biomass, such as acid, alkali, and pressurized hot water.
  • an enzyme composition for decomposing biomass is added to biomass, and the temperature is 40 ° C. to 100 ° C., the treatment pH is 3 to 7, and the biomass concentration is 0.1 to 30%. It is preferable to react. By setting to this range, it is possible to maximize the degradation efficiency of the enzyme composition for biomass degradation of the present invention.
  • the enzyme composition for decomposing biomass used in the method for producing a sugar liquid of the present invention can be recovered and further reused.
  • the endoxylanase mutant contained in the recovered enzyme composition for degrading biomass is 50% or more, 60% or more, 70% or more, or 80% or more, preferably 90, before being subjected to the sugar liquid production method. % Activity can be retained.
  • the enzyme composition for decomposing biomass can be collected by the following method. After adding the enzyme composition for biomass decomposition to biomass and performing a hydrolysis reaction, the hydrolyzate is solid-liquid separated.
  • the solution component obtained by solid-liquid separation includes the biomass decomposing enzyme composition and the sugar component, and the biomass decomposing enzyme composition and the sugar component are separated by filtration using an ultrafiltration membrane.
  • the molecular weight cut-off can pass through monosaccharides and oligosaccharides (disaccharides to 10 sugars). There is no limitation as long as it can be prevented.
  • the molecular weight cut off may be in the range of 2,000 to 50,000, and from the viewpoint of separating impurities that inhibit the enzyme reaction from the enzyme, more preferably the molecular weight cut off is 5,000 to 5,000.
  • the molecular weight is in the range of 50,000, more preferably in the range of 10,000 to 30,000 in the molecular weight cut-off.
  • Ultrafiltration membrane materials include polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, and sulfonated polyether.
  • PES polyethersulfone
  • PS polysulfone
  • PAN polyacrylonitrile
  • PVDF polyvinylidene fluoride
  • regenerated cellulose cellulose, cellulose ester
  • Sulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polytetrafluoroethylene and the like can be used, but it is preferable to use an ultrafiltration membrane made of a synthetic polymer such as PES or PVDF.
  • the endoxylanase mutant contained in the biomass degrading enzyme composition includes the position 35, position 44, position 62, It is more preferable to include an endoxylanase variant having the amino acid sequence of SEQ ID NO: 10 in which amino acids at 9 positions of position 63, position 101, position 102, position 61, position 65, and position 66 are substituted.
  • the sugar liquid obtained by the method for producing a sugar liquid of the present invention contains monosaccharide components such as glucose and xylose, it can be used as a raw sugar such as ethanol and lactic acid.
  • the sugar liquid obtained by the method for manufacturing a sugar liquid of the present invention contains xylooligosaccharide, xylobiose, xylotriose, etc., it can be used as an oligosaccharide for prebiotic applications, and is a human health food. Can be used as livestock feed.
  • Trichoderma reesei ATCC 66589 (distributed from ATCC) was inoculated to this preculture medium so as to be 1 ⁇ 10 5 cells / mL, and cultured at 28 ° C. for 72 hours with shaking at 180 rpm to prepare a preculture (shaking).
  • Apparatus BIO-SHAKER BR-40LF manufactured by TAITEC).
  • ATG in the nucleotide sequence CATATG at the NdeI site of pET11a is used as a translation start point as a methionine codon and contains a stop codon “TAG” at the 3 ′ end.
  • PET11a containing the nucleotide sequence of SEQ ID NO: 33 was cloned into BL21 (DE3) strain (Novagen).
  • the obtained recombinant BL21 (DE3) strain was cultured in LB medium containing ampicillin sodium 100 mg / L at 37 ° C. until OD600 became 0.6, and then isopropyl- ⁇ -D-1-thiogalactopyrano Sid (IPTG) 200 ⁇ M was added to induce expression of wild-type endoxylanase having the amino acid sequence of SEQ ID NO: 2. Expression was induced by incubating the medium at 16 ° C. for 20 hours, and then the recombinant BL21 (DE3) strain was collected by centrifugation at 4 ° C.
  • the collected cells were resuspended in Tris buffer pH 8 (20 mM Tris HCl, 50 mM NaCl).
  • the buffer containing the bacterial cells is completely frozen at -80 ° C for 1 hour and then thawed at room temperature for a total of 3 times to extract soluble proteins in the bacterial cells into the buffer solution. It was. Thereafter, the buffer solution was centrifuged at 18,000 rpm for 20 minutes at 4 ° C. to separate into a supernatant and cell residue.
  • the supernatant was passed through a Q-HP column (GE) pre-equilibrated with Tris buffer (20 mM, pH 8), the target endoxylanase was adsorbed on the column, and then eluted with a NaCl concentration gradient. .
  • GE Q-HP column
  • a solution having a NaCl concentration of 200 to 400 mM was collected. Thereafter, the endoxylanase fraction was further dialyzed against Tris buffer (20 mM, pH 8, 2M NaCl), and then passed through a Butyl HP column (GE) to adsorb endoxylanase.
  • Endoxylanase was eluted with a NaCl concentration gradient, and the fraction eluted with 1 M NaCl was collected. The fraction was further purified through a Superdex 200 16/60 gel filtration column (GE). The obtained purified endoxylanase was confirmed for impurities by SDS-PAGE.
  • Example 1 Preparation of DNA encoding endoxylanase mutant containing substitution of any one of position 35, position 44, position 62, position 63, position 101, position 102 and recombinant expression by E. coli
  • Production of a DNA encoding an endoxylanase mutant containing any one substitution of position 35, position 44, position 62, position 63, position 101, and position 102 was performed by the following procedure.
  • Table 1 shows pET11a containing a base sequence (SEQ ID NO: 33) encoding an amino acid sequence (SEQ ID NO: 2) excluding the signal peptide consisting of the N-terminal 34 amino acid residues from wild-type endoxylanase as a template (Comparative Example 1).
  • a DNA encoding an endoxylanase mutant having an amino acid substitution at a predetermined position was prepared by performing PCR using the described primer pair (Fw: forward primer, Rv: reverse primer). For the PCR, PrimeSTAR MaxDNA Polymerase kit (Takara Bio Inc.) was used. The primer pairs used are shown in Table 1 (SEQ ID NO: 14 to SEQ ID NO: 25).
  • the base sequence of the DNA encoding the obtained endoxylanase mutant (starting from the amino acid at position 35) is SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39. (Table 1).
  • the amino acid sequences of the respective endoxylanase mutants are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
  • the obtained DNA encoding the endoxylanase mutant was cloned into pET11a according to the procedure of Comparative Example 1. Next, using pET11a containing DNA encoding the endoxylanase mutant, the protein was expressed and purified according to the procedure of Comparative Example 1 to obtain each endoxylanase mutant.
  • Example 2 Production of DNA encoding endoxylanase mutant containing two substitutions at positions 35 and 62 and recombinant expression by Escherichia coli Endoxylanase mutation introduced in Example 1 with mutation introduced at position 35
  • pET11 containing DNA encoding the body SEQ ID NO: 3
  • SEQ ID NO: 18 and SEQ ID NO: 19 A DNA (SEQ ID NO: 40) encoding an endoxylanase variant with two substitutions into was generated.
  • the primer pairs used for this mutagenesis are the two types shown in Table 2.
  • the base sequence of the DNA encoding the produced endoxylanase mutant is shown in SEQ ID NO: 9.
  • the obtained DNA encoding the endoxylanase mutant was cloned into pET11a according to the procedure of Comparative Example 1.
  • the protein was expressed and purified according to the procedure of Comparative Example 1, and the endoxylanase mutant (SEQ ID NO: 9) of Example 2 was obtained. It was.
  • the nucleotide pairs of the DNAs encoding the primer pairs used and the obtained endoxylanase mutants are shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44 (Table 3).
  • the amino acid sequences of the respective endoxylanase mutants are shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 (excluding the starting methionine).
  • the obtained DNA encoding the endoxylanase mutant was cloned into pET11a according to the procedure of Comparative Example 1. Subsequently, pET11a containing DNA encoding the endoxylanase mutant was subjected to protein expression and purification according to the procedure of Comparative Example 1, and the mutant of Comparative Example 2 was obtained.
  • Example 3 Production of DNA encoding endoxylanase mutant containing nine substitutions at position 35, position 44, position 62, position 63, position 101, position 102, position 61, position 65, position 66 and by E. coli Recombinant expression
  • pET11 which was prepared in Example 2 and contains DNA (SEQ ID NO: 40) encoding an endoxylanase variant having two substitutions for cysteine at positions 35 and 62, respectively, position 44
  • a DNA encoding an endoxylanase mutant having a substitution at each position was prepared.
  • the primer pairs used for this mutagenesis are 9 types shown in Table 4.
  • the nucleotide sequence of the DNA encoding the prepared endoxylanase mutant is shown in SEQ ID NO: 41.
  • the amino acid sequence of the endoxylanase mutant is shown in SEQ ID NO: 10.
  • the obtained DNA encoding the endoxylanase mutant was cloned into pET11a according to the procedure of Comparative Example 1. Subsequently, pET11a containing DNA encoding the endoxylanase mutant was subjected to protein expression and purification according to the procedure of Comparative Example 1 to obtain the endoxylanase mutant (SEQ ID NO: 10) of Example 3.
  • Example 4 Endoxylanase activity measurement at each temperature of the endoxylanase mutant of Examples 1 to 3, the wild-type endoxylanase of Comparative Example 1 and the endoxylanase mutant of Comparative Example 2
  • the endoxylanase activity was 1% Measurements were made using birchwood xylan (Sigma Aldrich) as a substrate. Birchwood xylan was hydrolyzed by endoxylanase, and the amount of reducing sugar produced was measured by the dinitrosalicylic acid method (DNS) method using xylose as a standard.
  • DNS dinitrosalicylic acid method
  • 1 unit of endoxylanase activity was defined as the amount of enzyme required to produce 1 ⁇ mol of xylose from birchwood xylan at 50 ° C. for 1 minute, and the number of units was calculated. Moreover, based on each calculated unit value, the relative activity value was put together in Table 5 by making the activity value of 55 degreeC especially in a wild type (comparative example 1) into a reference
  • an endoxylanase mutant containing a substitution at a position selected from position 35, position 44, position 62, position 63, position 101, and position 102 exhibits higher activity even under high temperature conditions than wild type endoxylanase. It was clarified that the heat resistance was enhanced by the mutation. In particular, high heat resistance was observed in the endoxylanase mutants containing substitutions at positions 61, 65, and 66 in addition to positions 35, 44, 62, 63, 101, and 102.
  • Example 5 Method 1 for producing a sugar solution using an endoxylanase mutant An oligosaccharide liquid production was attempted using bagasse, which is a residue after sugarcane juice, as a raw material.
  • the endoxylanase the wild-type endoxylanase of Comparative Example 1 or the endoxylanase mutant of Example 3 was used.
  • immersion treatment was performed for 6 days in a 1N aqueous sodium hydroxide solution so that the biomass weight was 30% (w / w).
  • 0.5 g of the pretreated product was weighed into a 2 mL tube, and after adding water so that the final concentration of biomass became 10% (w / w), the pH was adjusted to 5 using diluted sulfuric acid.
  • Example 6 Production method 2 of sugar solution using endoxylanase mutant
  • Trichoderma-derived cellulase Reference Example 2
  • 10 mg / g-BM of Trichoderma-derived cellulase and 0.05 mg / g-BM of endoxylanase mutant (Example 3) were added to the composition adjusted to pH by the same method as in Example 5.
  • the reaction was carried out at 50 ° C. for 48 hours using a thermoblock rotator (SN-48BN manufactured by Nisshin Rika).
  • SN-48BN manufactured by Nisshin Rika
  • Example 7 Endoxylanase residual activity after reaction in Example 5 Using 500 Vl of the supernatant obtained after the reaction obtained in Example 5 using VIVASPIN500 (PES, molecular weight cut-off 10,000) (Sartorius) After ultrafiltration, the wild-type endoxylanase of Comparative Example 1 or the endoxylanase mutant of Example 3 was recovered. The recovered wild-type endoxylanase of Comparative Example 1 or the endoxylanase mutant of Example 3 and the wild-type endoxylanase of Comparative Example 1 diluted to the concentration at the time of xylan degradation (2.5 mg / l), the end of Example 3 The endoxylanase activity of the xylanase mutant was measured.
  • VIVASPIN500 PES, molecular weight cut-off 10,000
  • Endoxylanase activity was performed in the same manner as in Example 4 except that 1/10 of the collected endoxylanase or diluted endoxylanase was added and the reaction temperature was 50 ° C.
  • the diluted activity values of the wild-type endoxylanase of Comparative Example 1 and the endoxylanase mutant of Example 3 as the standard (100%), respectively, the recovered wild-type endoxylanase of Comparative Example 1 and the endoxylanase mutant of Example 3 were recovered.
  • the relative activity value was defined as the residual activity.
  • Table 8 shows the residual activity after recovery.
  • the endoxylanase mutant remained highly active after xylan degradation, confirming that it can be significantly reused for xylan degradation.
  • Example 8 Expression of wild-type endoxylanase and endoxylanase mutants by yeast Pichia pastoris Base encoding an amino acid sequence (SEQ ID NO: 2) excluding a signal peptide consisting of 34 amino acid residues at the N-terminus from wild-type endoxylanase Using pET11a containing the sequence (SEQ ID NO: 33) as a template (Comparative Example 1), PCR was performed using the primer pair of SEQ ID NO: 51 and SEQ ID NO: 53, whereby SEQ ID NO: 54, SEQ ID NO: 47, and SEQ ID NO: 55 were obtained. DNAs encoding in order were prepared.
  • PrimeSTAR MaxDNA Polymerase kit (Takara Bio Inc.) was used.
  • the base sequence of SEQ ID NO: 54 is upstream of the base sequence (SEQ ID NO: 48) encoding the endoxylanase variant (SEQ ID NO: 9) containing two substitutions at positions 35 and 62, and the base sequence of SEQ ID NO: 55 is downstream.
  • a DNA having the base sequence of SEQ ID NO: 54 added upstream and the base sequence of SEQ ID NO: 55 added downstream was artificially synthesized.
  • a competent cell of Pichia pastoris PPS-9010 strain (DNA2.0 company) was prepared.
  • a single colony of Pichia pastoris PPS-9010 strain in a 100 ml flask containing 20 ml of YPD liquid medium (yeast extract 1% (w / vol), peptone 2% (w / vol), glucose 2% (w / vol)) And incubating with shaking at 30 ° C. and 120 rpm for 16 hours (pre-culture).
  • the culture solution was transferred to a 50 ml falcon tube and collected by centrifugation at 500 ⁇ g for 15 minutes.
  • BEDS solution (10 mM bicine-sodium hydroxide buffer (pH 8.3), ethylene glycol 3% (vol / vol), dimethyl sulfoxide (DMSO) 5% (vol / vol), 9 ml of 1M sorbitol) was added and suspended gently, and 1 ml of 1M dithiothreitol (DTT) was further added, suspended gently, and shaken at 30 ° C. and 100 rpm for 5 minutes.
  • DTT dithiothreitol
  • the Falcon tube was cultured with shaking at 30 ° C. and 120 rpm for 1.5 hours, and 100 ⁇ L of the culture solution was added to a YPDS plate medium containing zeocin (1 mg / ml zeocin, yeast extract 1% (w / vol), peptone 2% (w / vol), glucose 2% (w / vol), 1M sorbitol, agar 2% (w / vol)) using a large rod and culturing at 30 ° C. for 2 to 3 days.
  • zeocin 1 mg / ml zeocin, yeast extract 1% (w / vol), peptone 2% (w / vol), glucose 2% (w / vol), 1M sorbitol, agar 2% (w / vol)
  • a colony having a large diameter was selected from the colonies generated on the plate, and each colony was inoculated and inoculated with a sterile toothpick into a 2 ml-deep 96-well plate with 0.5 ml of YPD liquid medium, and 4 at 28 ° C. and 1000 rpm. Cultured for days. The SDS-PAGE result of the culture supernatant is shown in FIG. It was confirmed that both proteins were secreted and expressed. In addition, since a band having a molecular weight larger than the molecular weight estimated from the primary structure was detected for the protein containing SEQ ID NOs: 2, 9, and 45, it was confirmed that sugar chain modification occurred in expression by Pichia.
  • Example 9 Activity comparison of endoxylanase variants Endoxylanase activity in the supernatants of various culture media obtained in Example 8 was measured. The activity was measured in the same manner as in Example 4 except that the supernatants of various culture solutions were added to 0.25 mg / mL and the reaction was performed at 50 ° C, 60 ° C, and 70 ° C. Based on the calculated unit values, the relative activity values are summarized in Table 11 with the activity value at 50 ° C. particularly in the wild type as a reference (100%). The supernatant containing the xylanase mutant retained high activity even under high temperature conditions. In addition, the enzyme expressed in yeast was found to have higher heat resistance as compared to the case expressed in E. coli in Example 4.
  • the endoxylanase mutant in the present invention exhibits high xylan degrading activity under high temperature conditions, it can be used for biomass hydrolysis, sugar solution production, and oligosaccharide production.

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Abstract

L'invention concerne une endoxylanase mutante présentant une stabilité thermique améliorée. L'invention concerne, donc, une endoxylanase mutante ayant une activité endoxylanase et comprenant une séquence d'acides aminés dans laquelle un ou plusieurs résidus d'acides aminés choisis au sein des positions correspondant aux positions 35, 44, 62, 63, 101 et 102 d'une séquence d'acides aminés de SEQ ID NO : 1 ont été substitués dans une séquence d'acides aminés d'une endoxylanase provenant de champignons filamenteux.
PCT/JP2015/081151 2014-11-05 2015-11-05 Endoxylanase mutante, composition enzymatique utilisable en vue de la décomposition d'une biomasse et procédé de production d'une solution de sucre WO2016072448A1 (fr)

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BR112017008702-2A BR112017008702A2 (pt) 2014-11-05 2015-11-05 ?mutante de endoxilanase, dna, vetor, célula, método de produção de mutantes de endoxilanase, composição enzimática e método de produção de solução de açúcar?
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CN111004789A (zh) * 2019-12-11 2020-04-14 云南师范大学 一种耐硫酸铵的木糖苷酶突变体v322dh328dt329e

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CN110904078B (zh) * 2019-12-11 2020-09-04 云南师范大学 一种耐硫酸钠和硫酸铵的木糖苷酶突变体v322r及其应用
CN111073876B (zh) * 2020-01-18 2021-07-27 江南大学 热稳定性提高的枯草芽孢杆菌脂肪酶a

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AU2015344324B2 (en) 2021-09-30
US10435728B2 (en) 2019-10-08
BR112017008702A2 (pt) 2018-02-27
AU2015344324A1 (en) 2017-04-27
CA2966330A1 (fr) 2016-05-12
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AU2015344324A2 (en) 2017-06-08
EP3216864A4 (fr) 2018-05-02

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